The present invention relates to a method and test composition for determination of an enzyme activity, and more particularly to a method for determination of an activity of an enzyme contained in a living body. The method comprises adding an appropriate substrate for an enzyme to a living body sample to thereby form a compound, which is hereinafter referred to as an enhancer, being capable of accelerating a rate of a reaction in which a chromogen is oxidized by an oxidase in the presence of oxygen; oxidizing the chromogen by the oxidase in the presence of the enhancer and oxygen to form a pigment; and quantitatively determining the pigment in a conventional manner. The present invention also pertains to a test composition suitable for carrying out such determination.
The enzyme to which the present invention is applicable includes, for example, .gamma.-glutamyl transpeptidase (.gamma.-GTP), leucine aminopeptidase (LAP), alanine aminopeptidase (AAP), cystine aminopeptidase (CAP), X factor as a coagulation factor, thrombin, plasmin of plasminogen series, kallikrein, chymotrypsin, alkali phosphatase, N-acetyl glucosaminase and amylase.
.gamma.-GTP, LAP and alkali phosphatase reflect troubles of biliary and hepatic organs. AAP and N-acetyl glucosaminase are an indicator for renal disorder. CAP is an indicator for movement in uterus of a pregnant woman. X factor as a coagulation factor, thrombin and plasmin of plasminogen series are involved in coagulation of blood. Kallikrein is an indicator for primary aldosteronism and hypertension. Chymotrypsin is an indicator for chronic pancreatitis. Amylase is an indicator for pancreatic disorder.
Heretofore, determination of the activity of .gamma.-GTP, LAP and AAP are conventionally carried out by adding a substrate for the enzyme to a sample containing the enzyme, further adding a necessary enzyme for deriving an analyzable substance from the substrate, if necessary, and measuring the rate of formation of a resulting measurable compound. For example, the activity of .gamma.-GTP or LAP is quantitatively determined by decomposing an appropriate substrate for .gamma.-GTP or LAP by the enzyme, thereby forming an aniline derivative, allowing the aniline derivative to react with a chromogen to thereby form a pigment, and quantitatively determining the rate of formation of the pigment. When the amount of the enzyme is small, a chromogen capable of forming a pigment having a high molecular extinction coefficient is used, but such a conventional method has a limit in determination of an activity of a trace amount of enzyme.